CN117980493A - Polysaccharide or polysaccharide mixture prepared from Paenibacillus polymyxa - Google Patents
Polysaccharide or polysaccharide mixture prepared from Paenibacillus polymyxa Download PDFInfo
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- CN117980493A CN117980493A CN202280060978.XA CN202280060978A CN117980493A CN 117980493 A CN117980493 A CN 117980493A CN 202280060978 A CN202280060978 A CN 202280060978A CN 117980493 A CN117980493 A CN 117980493A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/01—Hexosyltransferases (2.4.1)
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Abstract
The present invention relates to a polysaccharide or a polysaccharide mixture prepared from genetically modified production organisms for paenibacillus polymyxa, and to a method for preparing said polysaccharide or polysaccharide mixture.
Description
Technical Field
The invention relates to a polysaccharide or a polysaccharide mixture prepared from a genetically modified production organism of Paenibacillus polymyxa (Paenibacillus polymyxa).
Background
Polysaccharides are general biopolymers that occur in various life areas. They exist in large numbers in nature and accomplish a variety of tasks. In principle, polysaccharides can be classified into intracellular, structural and extracellular polysaccharides according to their function and/or location. Starch and glycogen are examples of intracellular polysaccharides and are effective energy storage polysaccharides in animals, algae or plants. Examples of structural polysaccharides are cellulose, chitin, xylan or mannan, which as a strong, hard structure impart mechanical strength to plants, insects or fungi. Extracellular polysaccharides include all polysaccharides secreted into the extracellular space. If the polysaccharide is completely secreted into the extracellular space and does not form a shell around the cell like the capsular polysaccharide, it is also called Extracellular Polysaccharide (EPS). However, the boundary between capsular and extracellular polysaccharides may not always be clear, as capsular polysaccharides sometimes bind only very loosely to the cell membrane, while extracellular polysaccharides may also be very close to the cell.
All polysaccharides consist of carbohydrate monomers. These monomeric sugars and their modifications form the basic structural unit (Grundbauteile) of the polysaccharide. Unlike plant heteropolysaccharides, microbial heteropolysaccharides are generally structurally regular and consist of repeating elements, so-called repeating units, having a uniform monomer sequence. Depending on the species and strain, these repeat units are mostly composed of two to eight sugar monomers. In extreme cases, the repeating unit may consist of up to fourteen sugar monomers. The synthesis of these repeat units and their attachment to the polysaccharide is regulated in microorganisms in so-called biosynthetic clusters, in which all enzymes required for synthesis are usually encoded.
The large number of different monomers and the large number of possibilities for their connection to each other make numerous polymer variants possible. Exopolysaccharides also have very diverse physical and chemical properties due to their high degree of structural variability. This makes them interesting materials with new properties or additives that impart the desired properties to the product.
One example of a known commercial EPS already existing on the market is xanthan gum. It is synthesized by Xanthomonas campestris (Xanthomonas campestris) and is used in particular in food technology as an emulsifier or foam stabilizer. However, xanthan gum is also used outside the food field. For example, it is used to adjust the viscosity of printer ink. Another polysaccharide that has been introduced into the U.S. and european union markets is gellan gum. This is EPS of the organism sphingomonas paucimobilis (Sphingomonas paucimobilis) which is capable of forming a thermoreversible gel and is therefore used as a gelling agent and stabilizer in food technology.
From a biotechnological point of view, such microbial polysaccharides are of particular interest, since they can be produced on a variable scale independently of the season and the place.
The gram-positive soil bacteria Paenibacillus polymyxa are known to produce polysaccharides under suitable fermentation conditions. In particular, polysaccharides produced by Paenibacillus polymyxa are known to be characterized by their high viscosity. This high viscosity enables the use of the polysaccharide as a rheological medium or binder in, for example, the food, cosmetic and/or pharmaceutical fields. On the other hand, the high viscosity of the produced polysaccharides is an obstacle to efficient production of polysaccharides, because polysaccharides impart high viscosity to the fermentation medium and thus make material transportation difficult and require increased energy input, for example, by stirring or heating.
Disclosure of Invention
The object of the present invention is to provide an efficient and energy-saving process for preparing polysaccharides which can be produced by Paenibacillus polymyxa.
By characterizing polysaccharides and elucidating their biosynthesis, a basis is provided for achieving the stated object. The results show that the polysaccharide produced by Paenibacillus polymyxa is a polysaccharide mixture of three individual polysaccharides, showing high viscosity only by the interaction of two of the polysaccharides. The inventors have elucidated the biosynthesis of each of these three individual polysaccharides.
The object according to the invention is achieved by mutagenesis, preferably directed mutagenesis, of individual genes of Paenibacillus polymyxa, which in turn enables the controlled production of low-viscosity single polysaccharides and/or low-viscosity mixtures thereof. The rheological properties of the polysaccharide and/or polysaccharide mixture can be adjusted by mixing them in suitable proportions and, for example, the properties of the polysaccharide mixture prepared from the wild type can also be achieved. Accordingly, the present invention relates to a process for the preparation and the various mutated production organisms used in the process for the preparation which are paenibacillus polymyxa, and the use of the polysaccharides or polysaccharide mixtures prepared according to the invention, for example as rheology media, binders, stabilizers, emulsifiers or flocculants, preferably in the food, pharmaceutical and/or cosmetic fields.
Drawings
Fig. 1 shows the structure of the repeat unit of Paenan I.
Fig. 2 shows the structure of the repeat unit of Paenan II.
Fig. 3 shows the structure of the repeat unit of PAENAN III.
Fig. 4 shows the monomer composition of the prepared polysaccharide. Carbohydrate fingerprints of EPS and mutant variants from Paenibacillus polymyxa DSM 365 were analyzed by HT-PMP method. Based on the ratio of monomers obtained and the detection of specific dimers in the MS analysis, the polymer composition was classified as the presence of different Paenan variants (I-III). The Δ epsO variant showed the same monomer composition as the wild-type polymer, but no pyruvic acid or the corresponding ketal was detected anymore.
FIG. 5 shows the viscosity curves of Paenan variants measured with cone-plate geometry at 20℃with (square ≡) and without (circular ≡) 0.5% sodium chloride at increasing shear rates from 0.01 to 1000/s. All measurements were performed three times and the error band shows the standard deviation.
FIGS. 6 and 7 show amplitude scans of 1% solutions of the indicated EPS variants produced by Paenibacillus polymyxa DSM 365 in aqueous solution and in the presence of 0.5% NaCl. G': storage modulus; g': loss modulus.
Figures 8 and 9 show frequency scans of 1% solutions of the indicated EPS variants produced by paenibacillus polymyxa DSM 365 in aqueous solution and in the presence of 0.5% NaCl. This variant was not measured in frequency sweeps due to the limited LVE range of PAENAN III under the conditions used. G' (circle): storage modulus; g "(square): loss modulus.
FIGS. 10 and 11 show temperature scans at 20-75℃of a 1% solution of the indicated EPS variants produced by Paenibacillus polymyxa DSM 365 in aqueous solution and in the presence of 0.5% NaCl. G' (circle): storage modulus; g "(square): loss modulus.
Fig. 12 shows a comparison of the monomer composition of Panean wild type with the monomer composition of the polysaccharide mixture after separate production and re-mixing.
Detailed Description
As described above, the present inventors have found that the polysaccharide produced by Paenibacillus polymyxa is a polysaccharide mixture composed of three single polysaccharides. These single polysaccharides are called Paenan I, paenan II and PAENAN III. The structures of the repeat units Paenan I, paenan II, and PAENAN III are shown in figures 1,2, and 3, respectively. The respective molecular weights are shown in table 4.
The biosynthesis of the single polysaccharide is illustrated and the following enzymes have been found to be necessary for the preparation of Paenan I: glycosyltransferase PepD and/or PepF, pyruvyl transferase EpsO and undecanopentenyl-glucose-phosphate transferase PepC. In the absence of the glycosyltransferase and the undecyipentenyl-glucose-phosphotransferase, no repeat unit is formed. Pyruvyltransferase EpsO allows ligation of a pyruvic acid residue to a terminal repeat unit. Such pyruvic acid residues are critical for gel-forming properties and the high viscosity of the resulting polysaccharide mixture.
The following enzymes are necessary for Paenan II preparation: glycosyltransferases PepT, pepU and/or PepV, and undecyipentenyl-glucose-phosphotransferase PepQ. In the absence of the glycosyltransferase and the undecyipentenyl-glucose-phosphotransferase, no repeat unit is formed. Furthermore Paenan II has GDP-L-fucose produced by GDP-L-fucose synthase. GDP-L-fucose synthase is encoded in gene fcl and gene fcl is only present once in the genome of Bacillus. Thus, generation of Paenan II can be suppressed by deleting fcl.
The following enzymes are necessary for PAENAN III preparation: glycosyltransferases PepI, pepJ, pepK and/or PepL, and undecanopentenyl-glucose-phosphate transferase PepC or PepQ. In the absence of one of the glycosyltransferase and the undecyipentenyl-glucose-phosphotransferase, no repeat unit is formed.
It was also found that the interaction between Paenan I and PAENAN III resulted in the formation of high viscosity and gel properties of the resulting polysaccharide mixture. In this regard, the pyruvate residue on the Paenan I repeat unit interacts with glucuronic acid in PAENAN III. Thus, if a polysaccharide mixture is to be produced after the production of individual polysaccharides, respectively, so that the gel properties and viscosity of Paenan wild-type are achieved, the function of EpsO in the production organism should be abandoned.
Thus, the polysaccharide mixtures Paenan I and Paenan II and the polysaccharide mixtures Paenan II and PAENAN III are also low-viscosity and can be produced simultaneously without the disadvantages of high-viscosity fermentation media.
These findings now enable mutagenesis, in particular directed mutagenesis, of the coding sequences of these enzymes, which leads to a loss of enzyme function and thus to production organisms capable of specifically producing low-viscosity single polysaccharides or low-viscosity polysaccharide mixtures. The low viscosity mono-polysaccharide or low viscosity polysaccharide mixture can be used, for example, as a functional binder for surface coatings, paints and in the food industry, for example in fruit juices or salad dressings and the like.
The desired beneficial properties of the polysaccharide mixture, such as adjustable viscosity and gel properties, can be recovered after separate production by mixing and optional heating. Thus, the method according to the present invention combines the advantages of efficient production of low viscosity mono-polysaccharides or low viscosity polysaccharide mixtures with the advantages provided by the high viscosity polysaccharide mixtures obtained after mixing low viscosity mono-polysaccharides or low viscosity polysaccharide mixtures by their broad applicability.
Thus, in one aspect, the present invention relates to a method for preparing a polysaccharide or a polysaccharide mixture by a production organism being paenibacillus polymyxa, wherein the method comprises at least one of the following steps, separate from each other:
(a) Preparing polysaccharide Paenan I by a producing organism for bacillus polymyxa, wherein at least one enzyme function of a polysaccharide biosynthetic cluster is turned off by genetic modification, thereby producing neither polysaccharide Paenan II nor polysaccharide PAENAN III; and/or
(B) Preparing polysaccharide Paenan II by a producing organism for bacillus polymyxa, wherein at least one enzyme function of a polysaccharide biosynthetic cluster is turned off by genetic modification, thereby producing neither polysaccharide Paenan I nor polysaccharide PAENAN III; and/or
(C) Preparing polysaccharide PAENAN III by a producing organism for bacillus polymyxa, wherein at least one enzyme function of a polysaccharide biosynthetic cluster is turned off by genetic modification, thereby producing neither polysaccharide Paenan I nor polysaccharide Paenan II; and/or
(D) Polysaccharides Paenan I and II are prepared by a production organism for bacillus polymyxa, wherein at least one enzyme function of the polysaccharide biosynthetic cluster is turned off by genetic modification so that polysaccharide PAENAN III is not produced; and/or
(E) Polysaccharides Paenan II and III are produced by a producing organism that is bacillus polymyxa, wherein at least one enzyme function of the polysaccharide biosynthetic cluster is turned off by genetic modification so that polysaccharide Paenan I is not produced.
The method according to the invention may further comprise purifying the produced polysaccharide or polysaccharide mixture after production.
The production organism modified by directed mutagenesis is Paenibacillus polymyxa. Paenibacillus polymyxa DSM 365 was used in the examples. However, all Paenibacillus polymyxa strains known to those skilled in the art to produce polysaccharides Paenan I, II, and III under suitable fermentation and/or culture conditions can be used.
The method according to the invention may further comprise, after the production and possible purification of the polysaccharide or polysaccharide mixture, mixing the produced polysaccharide or polysaccharide mixture, thereby obtaining a polysaccharide mixture having the following composition: a mixture of polysaccharides Paenan I and Paenan II, or a mixture of polysaccharides Paenan I and PAENAN III, or a mixture of polysaccharides Paenan II and PAENAN III, or a mixture of polysaccharides Paenan I, paenan II and PAENAN III.
As shown in example 5, at the time of mixing, the person skilled in the art can select the type of polysaccharide or polysaccharide mixture to be mixed, the mixing ratio and the concentration, in order to set the desired rheological properties of the obtained mixture. For example, by mixing Paenan I and PAENAN III in a ratio of 1:2m/v, a polysaccharide mixture with rheological properties similar to wild type Paenan can be obtained. However, it is also possible to produce mixtures having a higher or lower viscosity than, for example, the Paenan wild type. Those skilled in the art can adjust these properties as desired for the respective application.
Methods for targeted shut-down of gene function are known to those skilled in the art. In embodiments, the function of the described enzyme is turned off by CRISPR-Cas9 mediated knockout of the gene sequence encoding the corresponding enzyme. The basic procedure is described in Synth Biol (Oxf) by Hu tering et al; in month 1 of 2017. However, any method known to those skilled in the art for targeted shut-down of gene function may be used. For example, it is not absolutely necessary to delete the entire gene sequence. It is sufficient to delete only the region of the gene sequence responsible for the function of the gene. Genetic modifications resulting in the elimination of gene function may also be obtained by natural mutagenesis, e.g. mutations caused by UV radiation or chemical agents. Only a small effort is required to select strains mutated in this way according to the desired phenotype. Thus, genetic modification in the sense of the present invention is not limited to genetic engineering modification.
In order to obtain a production organism according to the invention, it may be sufficient to switch off only one function of the gene, as long as switching off the gene results in the mutated production organism no longer producing the undesired single polysaccharide. Specific embodiments with exemplary mutations are shown in the examples.
The method according to the invention may comprise selecting at least one enzyme function for turning off the polysaccharide biosynthetic cluster from among the genetic modifications that suppress the function of glycosyltransferases PepD and/or PepF and/or undecyipentenyl-glucose-phosphate transferase PepC so as not to produce polysaccharide Paenan I.
The method according to the invention may alternatively or additionally comprise a genetic modification for selecting at least one enzyme function for turning off polysaccharide biosynthesis clusters from among the genetic modifications suppressing the functions of glycosyltransferases PepT, pepU and/or PepV and/or undecenyl-glucose-phosphotransferase PepQ and/or GDP-L-fucose synthase such that polysaccharide Paenan II cannot be produced.
The method according to the invention may alternatively or additionally comprise selecting at least one enzyme function for turning off the polysaccharide biosynthesis cluster from among the genetic modifications that suppress the function of glycosyltransferases PepI, pepJ, pepK and/or PepL so as not to produce polysaccharide PAENAN III.
An exemplary genetic modification that turns off at least one enzymatic function of the polysaccharide biosynthetic cluster, thereby producing neither polysaccharide Paenan II nor polysaccharide PAENAN III is selected from the group consisting of genetic modifications that suppress: the functions of GDP-L-fucose synthase, or glycosyltransferases PepI, pepT, pepU and PepV, or glycosyltransferases PepK, pepT, pepU and PepV, or glycosyltransferases PepL, pepT, pepU and PepV, or glycosyltransferases PepT and PepL.
An exemplary genetic modification that turns off at least one enzyme function of the polysaccharide biosynthetic cluster, thereby producing neither polysaccharide Paenan I nor polysaccharide PAENAN III is selected from the group consisting of a genetic modification that suppresses the function of glycosyltransferases PepF and PepJ or the function of glycosyltransferases PepD and PepJ.
An exemplary genetic modification that turns off at least one enzyme function of the polysaccharide biosynthetic cluster, thereby producing neither polysaccharide Paenan I nor polysaccharide Paenan II is selected from the group consisting of genetic modifications that suppress the function of undecyipentenyl-glucose-phosphotransferase PepQ and glycosyltransferase PepF.
The invention also relates to a composition comprising polysaccharide Paenan I, but neither polysaccharide Paenan II nor polysaccharide PAENAN III; or alternatively
A composition comprising polysaccharide Paenan II, but neither polysaccharide Paenan I nor polysaccharide PAENAN III; or alternatively
A composition comprising polysaccharide PAENAN III, but neither polysaccharide Paenan I nor polysaccharide Paenan II; or alternatively
A composition comprising polysaccharide Paenan I and polysaccharide Paenan II, but not comprising polysaccharide PAENAN III; or alternatively
A composition comprising polysaccharide Paenan II and polysaccharide PAENAN III, but not comprising polysaccharide Paenan I.
The invention also relates to a production organism which is a paenibacillus polymyxa in which at least one enzyme function of the polysaccharide biosynthesis cluster is turned off by genetic modification, so that polysaccharides Paenan II and PAENAN III cannot be produced; or alternatively
A production organism that is a paenibacillus polymyxa in which at least one enzyme function of a polysaccharide biosynthetic cluster is turned off by genetic modification, thereby failing to produce polysaccharide Paenan I and polysaccharide PAENAN III; or alternatively
A production organism that is a paenibacillus polymyxa in which at least one enzyme function of a polysaccharide biosynthetic cluster is turned off by genetic modification, thereby failing to produce polysaccharide Paenan I and polysaccharide Paenan II; or alternatively
A production organism that is a paenibacillus polymyxa in which at least one enzyme function of a polysaccharide biosynthetic cluster is turned off by genetic modification, thereby failing to produce polysaccharide PAENAN III; or alternatively
A production organism which is a paenibacillus polymyxa in which at least one enzyme function of a polysaccharide biosynthetic cluster is turned off by genetic modification, thereby failing to produce polysaccharide Paenan I.
The polysaccharides or polysaccharide mixtures produced by the production organisms according to the invention with the method according to the invention can be used widely because of their advantageous properties which may already be present on the one hand when using the polysaccharides produced according to the invention or the polysaccharide mixtures produced according to the invention themselves or which may be obtained when mixing the polysaccharides produced according to the invention or the polysaccharide mixtures produced according to the invention with further polysaccharides produced according to the invention or further polysaccharide mixtures produced according to the invention.
Thus, the invention also includes the use of a polysaccharide comprising Paenan I, paenan II or PAENAN III, or a mixture of any combination of Paenan I, paenan II and PAENAN III, as a rheology, binder, stabilizer, emulsifier or flocculant, preferably in the food, pharmaceutical and/or cosmetic field.
In particular, the polysaccharide comprising Paenan I, paenan II, or PAENAN III, or a mixture of any combination of Paenan I, paenan II, and PAENAN III, may be used as an additive to a food product, such as, but not limited to, a particle selected from baked goods, soups, sauces, mayonnaise, ketchup, jams, citrus sauces, jellies, cans (fruits and vegetables), salad dressings, ice cream, mixed milk beverages, puddings, salted vegetables, can meats, and fish, or as an additive in cosmetic products, such as, but not limited to, a shower gel, a cosmetic cream, and emulsion, a shampoo, a skin cream, a toothpaste, a moisturizing cream, or as an additive in pharmaceutical or medical products, such as, but not limited to, a particle selected from wound dressing, a particle for controlled release of active ingredients, eye drops, a tablet coating, a capsule for nutritional supplements, or in tissue engineering to support surface adhesion after surgery, in water injection for petroleum extraction, in bioremediation and wastewater purification, for heavy metal binding, as a cement additive to maintain particles during curing, or as a paint additive.
The method according to the invention, as well as the production organism according to the invention and the use according to the invention are now described in the following by way of example.
Examples
EXAMPLE 1 preparation of mutant production organisms
Paenibacillus polymyxa DSM 365 was purchased from the German collection of microorganisms and cell cultures (DSMZ, germany). Coli NEB Turbo cells (new england biology laboratory, usa) were used for plasmid construction. Coli S17-1 (DSMZ strain DSM 9079) was used for transformation of Paenibacillus polymyxa DSM 365 by conjugation. The strains produced are listed in Table 1.
Table 1: list of production organisms prepared
All knockouts were performed as described previously (Tu tering et al, 2017). Briefly, the gRNA of each target was cloned into plasmid pCasPP by portal assembly using BbsI. Thereafter, 1kb upstream and downstream homologous flanks of the gene of interest were ligated into a unique SpeI site, followed by transformation of chemically competent E.coli S17-1. Coli S17-1 having different plasmids was used to transform Paenibacillus polymyxa by conjugation. Overnight cultures of donor and recipient strains were diluted 1:100 with selective and non-selective LB medium and incubated at 37℃for 3 hours at 280 rpm. 900. Mu.l of the recipient culture was subjected to heat shock at 42℃for 15 minutes and mixed with 300. Mu.l of the donor strain. Cells were centrifuged at 6,000Xg for 2 min, resuspended in 800. Mu.l LB medium and added dropwise to non-selective LB agar plates. After 24 hours incubation at 30℃the cells were scraped, resuspended in 500. Mu.l LB broth and 100. Mu.l of these were plated on selective LB agar containing 50. Mu.g/ml neomycin and 20. Mu.g/ml polymyxin for counter selection. After 48 hours of incubation at 30℃the successful transformation of the Paenibacillus polymyxa adapter was analyzed by colony PCR. The confirmed knockout strain was subjected to plasmid digestion by culturing in LB broth without antibiotic selection pressure and subsequent replica plating on LB agar plates with and without neomycin. Strains that did not grow on plates with selectable markers were verified by target region sequencing and used for further experiments. All plasmids and oligonucleotides used to obtain the knockout strain are listed in tables 2 and 3 below
Table 2: some plasmids were used and prepared.
Table 3: some of the oligonucleotides used. The overhang for the golden gate assembly is shown in lowercase letters. Restriction sites for cloning are underlined. For each KO construct, two sets of sgrnas were designed, tested, and listed below if a successful knockout was made
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EXAMPLE 2 fermentative production of polysaccharide
Unless otherwise indicated, all media components were purchased from Carl Roth GmbH (Germany). For the cloning method, the strain was cultivated in LB medium (5 g/L yeast extract, 10g/L tryptone, 10g/L NaCl) and additionally had 50. Mu.g/mL neomycin and 20. Mu.g/mL polymyxin. Make-up is performed if necessary. All strains were stored in 30% glycerol at-80 ℃. Prior to cultivation, the strain was streaked onto LB agar plates and allowed to grow at 30℃for 24 hours.
The preparation of the fermentation medium containing 30g/L glucose, 0.05g/L CaCl 2 x 2H2 O, 5g/L tryptone, 1.33g/L MgSO 4 x 7H2O、1.67g/l KH2PO4, 2ml/L RPMI 1640 vitamin solution (Merck, germany) and 1ml/L microelement solution (2.5g/l FeSO4、2.1g/l C4H4O6Na2 x 2H2O、1.8g/l MnCl2 x 4H2O、0.258g/l H3BO3、0.031g/l CuSO4 x 5H2O、0.023g/lNaMoO4 x 2H2O、0.075g/l CoCl2 x 7H2O、0.021g/l ZnCl2). preculture was identical to that of the fermentation medium except that the glucose concentration was reduced to 10g/L and 20g/L MOPS was additionally added to buffer to pH 7.
The fermentative production of EPS was carried out in a 1L bench top DASGIP parallel bioreactor system (Eppendorf, germany) with a working volume of 500ml, equipped with a 6-blade Rushton impeller, with a controlled pH of 6.8 and a pO 2 saturation of 30%. Batch culture was started at an initial OD600 of 0.1 by inoculating the preculture at the appropriate volume. After fermentation, the biomass was separated by centrifugation (15,000Xg, 20 ℃ C., 20 minutes) and the supernatant was then subjected to cross-flow filtration using a 100kDa filter cartridge (Hydrost, sartorius AG, germany). High viscosity EPS variants, such as those produced by the wild type, are diluted with ddH 2 O1:10 prior to centrifugation. The concentrated supernatant was then slowly poured into twice the volume of isopropanol. The precipitated EPS was collected and dried in a VDL53 vacuum oven (Binder, germany) at 40 ℃ overnight. The dry weight of the EPS obtained was determined gravimetrically before grinding it into a fine powder (Mixer Mill MM400, retsch GmbH, germany) in a ball Mill for 1 minute at 30 Hz.
EXAMPLE 3 characterization of the polysaccharide produced
The monomer composition of the EPS variants produced was analyzed by the 1-phenyl-3-methyl-5-pyrazolone high throughput method (HT-PMP) (Hu hmann, schmid & Sieber, 2014). Briefly, a 0.1% EPS solution was hydrolyzed in a 96-well plate, sealed with a silica gel pad, and further covered with a custom-made metal device with 2M TFA (90 min, 121 ℃). The sample was neutralized with 3.2% NH 4 OH. Mu.l of PMP master mix (0.1M methanolic PMP:0.4% ammonium hydroxide 2:1) was added to 25. Mu.l of the neutralized hydrolysate and incubated for 100 min at 70℃in a thermal cycler. Mu.l of the derivatized sample was mixed with 25. Mu.l of 0.5M acetic acid and 125. Mu.l of ddH 2 O, filtered with a 0.2 μm filter plate (1,000Xg, 2 minutes) and then subjected to HPLC-UV-MS using a Ultimate 3000RS HPLC system (Dionex, USA). The separation was carried out on a reverse phase column (Gray C18, 100x 2mm,1.8 μm particle size, macherey-Nagel, USA) set at 50 ℃. Gradient elution was performed using mobile phase A (5 mM ammonium acetate, pH 5.6, containing 15% acetonitrile) and mobile phase B (100% acetonitrile) at a constant pump rate of 0.6 mL/min.
To detect the presence of a single Paenan polymer, a carbohydrate fingerprint was determined for each variant (fig. 4). A monomer ratio of 3:1:1:1 (Glc: man: gal: pyr) is expected for Paenan I, while a Glc: gal: glcA: fuc equimolar ratio is expected for Paenan II, and a monomer ratio of 2:2:1 (Glc: man: glcA) is expected for PAENAN III. Because uronic acid is highly susceptible to degradation during chemical hydrolysis, glcA is greatly underestimated for all uronic acid containing polysaccharide compositions. In addition to monomer composition, previously identified key dimers detected by MS/MS analysis were also used to assign a combined knockout variant to each Paenan variant. By assigning the pyruvate ketal present to Paenan I, glcA-Fuc dimer to Paenan II, and GlcA-Man dimer to PAENAN III, the polymer composition of each knockout variant was determined while maintaining the natural polysaccharide profile in these variants. From now on, each EPS variant will be named according to the existing Paenan variant, not according to the gene deletion that leads to the respective phenotype.
The molecular weight of the Polymer variants was determined by size exclusion chromatography using the Agilent 1260Infinity system (Agilent Technologies, germany) equipped with refractive index detector (SECcurity GPC 1260) and SECcurity SLD7000 seven-angle static light scattering detector (PSS Polymer STANDARDS SERVICE, germany). To this end, 0.5g/l of each variant was reconstituted in 0.1M LiNO 3 and 100. Mu.l of sample was injected into the system at 30 minute intervals and kept at 50℃with a TSKgel SuperMP (PW) -H pre-column and two consecutive TSKGel SuperMultipore PW-H columns (6.0mm ID x 15cm,TOSOH Bioscience, germany). As eluent, 0.1M LiNO 3 was used, with a constant flow rate of 0.3ml/min. Absolute molecular weights were determined by light scattering and polymer concentration and further cross-validated with 12-point-pullulan standards (384 Da-2.35 MDa) and 4.5MDa xanthan gum references (table 4).
Table 4: the calculated molecular weight of the Paenan variant was obtained by GPC analysis with the aid of a 0.5% eps solution in 0.1M LiNO 3.
Although there are three different polymers in wild-type EPS, it is not possible to isolate the individual Paenan variants clearly. Analysis of each Paenan variant showed that Paenan I and PAENAN III had similar molecular weight distributions. Only Paenan II appears to be significantly smaller, with a size of 5.5.105 Da. Given the lower proportion of Paenan II in wild-type polymer, this may explain why previous attempts to analyze the exopolysaccharide of paenibacillus polymyxa DSM 365 failed to distinguish between multiple Paenan variants. Interestingly, although the depropionylated polymer also showed a small peak at about 3.0.106 Da, a significantly greater major molar mass was detected as 8.8.106 Da compared to all other Paenan variants. In contrast to the production of xanthan gum with side chains irregularly having acetyl or pyruvyl residues, all of the repeat units of Paenan I appear to be modified by pyruvic ketals. Thus, loss of this feature in the Δ epsO knockout variant may affect chain length control in paenibacillus polymyxa, which leads to increased molecular weight and other rheological properties. Or pyruvylation may also affect the hydrodynamic radius of the polymer, thereby affecting SEC-MALS analysis.
Example 4 rheological Properties
A1% (w/w) solution of each polymer in ddH 2 O and 0.5% NaCl (85 mM) was prepared for rheological analysis. Conductivity of each solution was measured using an LF413T-ID electrode (Schott Instruments, germany) to determine residual salt concentration of the fermentation medium. Rheological measurements were performed using a pressure controlled rotational rheometer MCR 300 (Anton Paar, austria) equipped with a CP 50-1 cone plate measurement system (diameter 50mm, cone angle 1 °, measurement gap 50 μm). All measurements were performed at 20 ℃ with the exception of temperature scanning, controlled by TEK 150P temperature unit. After the solution was applied to the rheometer, all samples were incubated at 20 ℃ for 5 minutes before starting the measurement. All experiments were performed in triplicate in technology.
The viscosity profile was measured at a shear rate increasing logarithmically from 10 -3/s to 10 3/s by: every 10 times 3 data points, the measurement time for each data point is reduced from 100-5 seconds.
The amplitude sweep is measured at a frequency of 1Hz with a shear stress amplitude that increases logarithmically from 10 -1 to 10 3 Pa.
The frequency sweep is performed in the linear viscoelastic region (LVE) with a frequency that increases logarithmically from 10 -2 to 10 Hz.
Temperature scans were performed in the LVE at a frequency of 1Hz, with the temperature used rising from 20 ℃ to 75 ℃ and the heating rate being 4 ℃/min. The edges of the cone-plate measuring system are covered with low viscosity paraffin oil (Carl Roth, germany) to prevent evaporation.
Thixotropic properties were assessed by a three-stage oscillatory shear sequence. In the first stage, the sample is subjected to shear stress in the LVE region and then to high oscillatory shear of 10 3 Pa for 30 seconds. The 10 minute recovery of structure was then measured in LVE.
Studies of flow characteristics showed that all Paenan variants exhibited universal shear thinning behavior (FIG. 5). The highest viscosity and shear thinning properties were observed for the Paenan wild type (I & II & III) with NaCl added, followed by the Paenan wild type without monovalent cations. All individual variants Paenan I-III do not exhibit significantly high viscosities and exhibit flow characteristics that approximate newtonian fluids over a moderate shear rate range. Only Paenan wild type and Paenan I & III showed strict shear thinning properties and high viscosity according to the power law, which increased in the presence of 0.5% NaCl. In the case of the wild-type Paenan desuvylate, the viscosity drops drastically and the addition of NaCl now has a detrimental effect, similar to the desuvylate of xanthan. In the case of xanthan gum, this effect can be attributed to lower cation-mediated intermolecular interactions between the individual polymer molecules. Data from different Paenan polymers indicated similar effects between Paenan I and PAENAN III molecules. The Paenan I & III combination showed almost the same properties as the Paenan I & II & III combination, which highlights the interaction between Paenan I and III as the main reason for the high viscosity of the polymer. This interaction can be explained by the cation mediated interaction of the Paenan I terminal pyruvate residue with the negative charge of glucuronic acid in the PAENAN III backbone. Interestingly, none of the combinations Paenan I & II and Paenan II & III produced wild type character. Thus, glucuronic acid in Paenan II backbone does not appear to interact with pyruvoyl groups of Paenan I, as shown by the low viscosity of this combination. This is particularly interesting because the single side chain of Paenan II suggests that the charge in Paenan II is more accessible than the two side chains of PAENAN III that bind glucuronic acid and adjacent mannose (fig. 1-3). There are two explanations for the interactions of the individual molecules. First, the formation of a single co-helix of Paenan I or II or III molecules and the subsequent interaction of these helices. Second, the formation of the isospiral of Paenan I & II & III. Studies of the individual polymers provide evidence for the latter. If, as is hypothesized, the pyruvylated side chains protrude outwards, as in xanthan gum, the formation of the homohelices also suggests a strong interaction between the homohelices of Paenan I alone. On the other hand, if Paenan I forms a homospiral, the inwardly protruding side chains will give a structure similar to that described for the diutan and may also be the cause of the viscosity decrease. In contrast to diutan, the backbone of Paenan I is not negatively charged due to the lack of uronic acid in the backbone. In all cases, the differences in Paenan II and PAENAN III helical arrangement can explain the strong interactions of Paenan I & III, but not the interactions between Paenan I & II and Paenan II & III, which led to further investigation of secondary and tertiary polymer structures.
Detailed studies of individual polymer variants and combinations of Paenan II & III showed multiple structural adhesive regions, which are represented by up to three different K and n values in the power law of a single cross section (table 5). This phenomenon is present for Paenan I and Paenan II alone and for the combination of Paenan I & II and Paenan I & III. However, only a single structural adhesive region was observed for PAENAN III, paenan I & III or wild type compositions. In Paenan I & II, the Newton region was more pronounced in the presence of NaCl, which may be the reason for the slight appearance of the Newton region in the presence of NaCl in Paenan wild-type and Paenan I & III. Thus, this effect can be attributed to Paenan I or Paenan II.
Table 5: model parameters of power law fitting of different Paenan combinations with and without 0.5% NaCl added (K, n). When fitting multiple sections separately, the K and n values for each section are shown in ascending order of the corresponding shear rate range.
* : Having flow characteristics of Newtonian fluid
The basic viscoelastic properties determined by amplitude scanning (fig. 6 and 7) are listed in table 6. Paenan wild type (I & II & III) exhibited a soft and elastic gel-like character with a yield strength of 9.1Pa (32% elongation), a yield point of 51.1Pa (550% elongation) and a damping coefficient within LVE of 0.3. The addition of 0.5% NaCl resulted in an increase in yield strength (Ausbeute) and yield point of 32.3Pa and 90.8Pa, corresponding to elongation of 82% and 590%. The damping factor of 0.1 and the apparent G "peak after LVE indicate stronger but more brittle gel properties. Frequency scanning (fig. 8) also shows the viscoelastic, fluid-like properties of G 'and G "of Paenan I & II & III, mainly elastic properties throughout the frequency range studied, which with the addition of NaCl goes more towards gel-like properties with lower frequency dependence of G' and G". No apparent crossover points at low frequency, with or without NaCl added, indicating long term stability of the network.
This gel property is likely caused by a cation-mediated interaction between the pyruvoyl group of Paenan I and the-COO-group of glucuronic acid of PAENAN III. Depropionylation of Paenan I & II & III, which resulted in a complete loss of viscoelastic properties, provides further evidence for this. In addition, all individual Paenan polymers and mixtures of Paenan I & II and Paenan II & III were shown to have main fluid properties similar to max Wei Texing (fig. 8 and 9). Interestingly, the typical transition point of max Wei Liuti was not observed at higher frequencies in the presence of NaCl, except Paenan II, and the data indicated that G' began to drop at higher frequencies, which resulted in fluid properties at low and high frequencies. This is particularly the case with Paenan I & III.
The high viscosity and the pronounced intermolecular network result in gel-like character, making the polymer variant an interesting compound as a rheological thickener. Like other microbial polysaccharides, potential applications as rheology modifiers in foods and beverages, and technical applications such as oil drilling, appear to be promising. The viscosity increasing effect was greatly enhanced compared to these polysaccharides, indicating that lower EPS concentrations were required to achieve similar results. In addition, structurally related polysaccharides from paenibacillus polymyxa 2H2 have recently shown excellent compatibility with commonly used surfactants (such as lauryl sulfate or cocamidopropyl betaine), which are commonly used in cosmetic and care products. Thus, the use of wild-type EPS compositions containing Paenan I & II & III paenibacillus polymyxa DSM 365 is suitable as a sustainable thickener for a variety of applications, which can replace commercially available petroleum-based acrylic compounds.
Table 6: viscoelastic properties of Paenan polymer variants. n.b.: because there is no measurement uncertainty
The Paenan I & III mixture exhibited very similar gel-like properties to Paenan I & II & III, indicating that interactions exist primarily between Paenan I and PAENAN III. However, paenan I & III exhibited lower gel strength compared to Paenan I & II & III, with and without 0.5% NaCl added, yield strengths of 13.9Pa and 35.8Pa, and the G "peak at the end of the LVE region was less pronounced in the presence of NaCl. This suggests that these polymers interact poorly. Studies of the amplitude scans of the individual polymers showed that Paenan I and II each exhibited viscoelastic, fluid-like properties, whereas PAENAN III exhibited only purely fluid properties. Without the formation of a gel-like network, the addition of NaCl resulted in a decrease in G' and G ", whereas Paenan I had a higher salt stability than Paenan II. This also becomes evident in the Paenan I & II mixture, where the effect of NaCl is more comparable to Paenan I than Paenan II. These effects indicate an interaction between Paenan I and II, which may be responsible for the increased gel strength compared to Paenan I & III, paenan I & II & III. Since both Paenan II and PAENAN III have glucuronic acid in the backbone, the strong interaction of Paenan I & III suggests that glucuronic acid of PAENAN III has better accessibility than glucuronic acid of Paenan II. In contrast, interactions between Paenan II and PAENAN III may lead to other structural arrangements of these polymers, which lead to better accessibility of glucuronic acid in Paenan II, leading to enhanced interactions between Paenan I & II in the polymer mixture.
In contrast to the natural polysaccharide compositions containing Paenan I & II & III, the absence of a single polymer results in a significant change in viscoelastic properties. While the Paenan I & III combination always also shows a unique intermolecular network that produces gel-like properties, each biopolymer shows liquid-like properties that always also form a film after drying. Thus, the use of wild-type EPS compositions is significantly different. Thus, one application of the polysaccharides of the present invention is in the formation of edible films and packaging materials similar to those having pullulan. In another aspect, the polysaccharides of the present invention may be used in high value biomedical applications as coating materials in pharmaceutical systems for controlled drug release. For other charged polysaccharides, such as hyaluronic acid and alginate, chemical modification of the functional groups improves targeting to specific cell types, which enables an efficient drug delivery system. In addition, polysaccharides produced by other Paenibacillus polymyxa strains exhibit antioxidant activity, which may further improve pharmacological applications.
Temperature scans showed that the viscoelastic properties of Paenan I & II & III, with or without NaCl, were highly temperature dependent (fig. 10 and 11). The viscoelastic properties of Paenan I & III show an even higher temperature dependence, which can be reduced by adding NaCl. Although the natural polysaccharide composition containing all three Paenan variants retained weak gel properties up to 75 ℃ in the presence of NaCl, the deletion of Paenan II resulted in a loss of temperature stability. This suggests that Paenan II has a stabilizing effect on the temperature stability of the polymer network, which is also evident by the high temperature stability of Paenan II compared to Paenan I & III. For Paenan II, an increase in G' and G "can be observed during the temperature rise, which is also enhanced by the addition of NaCl. This effect is similar to that observed in the case of the unmodified xanthan variant described previously, and can also be explained by structural rearrangement of the single polymer. However, none of these effects occurred for Paenan II in any other combination with the other Paenan polymers, indicating a different arrangement of the individual polymers in the mixture, which highlights the differences between Paenan I & II & III and Paenan I & III in their viscoelastic properties described previously.
Table 7: temperature stability of the different paenan variants. n.b.: is not determined when it cannot be measured in LVE range
In addition, thixotropic properties were determined by a three-stage oscillating shear stress test (table 8). Although structural recovery was observed in all combination variants of Paenan, after three minutes of lossless shear loading, the wild-type EPS mix measured only 86.8% of the initial gel strength. This highlights the unique intermolecular network that requires more time to recover and coordinate the non-covalent interactions between the individual polymers. For polysaccharide compositions with Paenan I & III, a similar delayed structure recovery effect was observed, confirming the hypothesis that the gel-like character is mainly derived from cation-mediated interactions between pyruvic acid of Paenan I and glucuronic acid residues of PAENAN III. In contrast, for all other knockout variants, immediate recovery of the structure was observed, which resulted in initial gel strength. Thus, the different variants can be used as binders for imparting thixotropic properties to paints and coatings commonly used with different rheological properties.
Table 8: thixotropic recovery of Paenan variants after the three-stage oscillatory shear test. The thixotropic recovery after 30/90/180s was calculated from G' relative to the initial value determined in the LVE zone. n.b.: the variant was not defined due to the limited range of LVE
Rheological properties of the exopolysaccharide produced by paenibacillus polymyxa DSM 365 were characterized using CRISPR-Cas9 mediated glycosyltransferase knockout. The viscoelastic properties of each Paenan variant and combinations thereof were analyzed in detail. While wild-type EPS compositions exhibit high viscosity and gel-like properties, the knockout variants exhibit significantly altered physicochemical properties that depend on the individual polysaccharides present. Thus, the different polysaccharide compositions may be used in a wide range of applications, for example as thickeners or coating materials.
EXAMPLE 5 mixing of separately prepared polysaccharides and/or polysaccharide mixtures
As shown in fig. 12, the monomer composition of wild-type Paenan and its rheological properties can be restored by mixing separately prepared polysaccharides. The strong gel properties of the wild-type polymer composition are based on the interaction of the pyruvate group of Paenan I (PI) and GlcA of PAENAN III (PIII). By mixing the low viscosity single polymers PI and PIII in a 1:2 ratio, the naturally occurring monomer composition of the deletion variant Δ pepQTUV can be reconstituted and thus the gel formation and high viscosity polymer properties can be restored (see also table 8).
Table 8 shows the viscosities of exemplary polysaccharide mixtures obtained by mixing separately prepared polysaccharides at 1:2.
* : At a shear rate of 1.04/s
* *: In the linear viscoelastic range
Δ pepI refers to a mutation by which a producing organism carrying the mutation cannot reproduce PAENAN III, but only Paenan I and Paenan II. Thus, by mixing with PAENAN III, a mixture similar to the Wild Type (WT) can be produced. These results show that by selecting a single polysaccharide or polysaccharide mixture, mixing ratio and total EPS concentration, a desired viscosity range can be set which can correspond to the Paenan wild-type viscosity, but can also be lower or higher than the wild-type viscosity. This represents flexibility and diversity of applications.
Claims (12)
1. A process for preparing a polysaccharide or a polysaccharide mixture from a production organism which is bacillus polymyxa, wherein the process comprises at least one of the following steps, separate from each other:
(f) Preparing polysaccharide Paenan I from a production organism that is bacillus polymyxa, wherein at least one enzyme function of a polysaccharide biosynthetic cluster is turned off by genetic modification, thereby producing neither polysaccharide Paenan II nor polysaccharide PAENAN III; and/or
(G) Preparing polysaccharide Paenan II from a production organism that is bacillus polymyxa, wherein at least one enzyme function of a polysaccharide biosynthetic cluster is turned off by genetic modification, thereby producing neither polysaccharide Paenan I nor polysaccharide PAENAN III; and/or
(H) Preparing polysaccharide PAENAN III from a production organism that is bacillus polymyxa, wherein at least one enzyme function of a polysaccharide biosynthetic cluster is turned off by genetic modification, thereby producing neither polysaccharide Paenan I nor polysaccharide Paenan II; and/or
(I) Preparing polysaccharides Paenan I and II from a production organism that is bacillus polymyxa, wherein at least one enzyme function of a polysaccharide biosynthetic cluster is turned off by genetic modification so that polysaccharide PAENAN III is not produced; and/or
(J) Polysaccharides Paenan II and III are produced from a production organism that is a paenibacillus polymyxa, wherein at least one enzyme function of the polysaccharide biosynthetic cluster is turned off by genetic modification so that polysaccharide Paenan I is not produced.
2. The process according to claim 1, wherein the polysaccharide or polysaccharide mixture prepared is purified after preparation; and/or wherein the producing organism is Paenibacillus polymyxa DSM365.
3. The method according to claim 1 or 2, wherein the prepared polysaccharide or polysaccharide mixture is mixed, thereby obtaining a polysaccharide mixture having the following mixture: a mixture of polysaccharides Paenan I and Paenan II, or a mixture of polysaccharides Paenan I and PAENAN III, or a mixture of polysaccharides Paenan II and PAENAN III, or a mixture of polysaccharides Paenan I, paenan II, and PAENAN III.
4. A method according to claim 3, wherein Paenan I and PAENAN III are mixed in a ratio of 1:2 m/v.
5. The method according to any one of claims 1 to 4, wherein the genetic modification that causes at least one enzyme function of the polysaccharide biosynthetic cluster to be shut down so as not to produce polysaccharide Paenan I is selected from the group consisting of a genetic modification that suppresses the function of glycosyltransferase PepD and/or PepF and/or undecyipentenyl-glucose-phosphate transferase PepC, and/or wherein the genetic modification that causes at least one enzyme function of the polysaccharide biosynthetic cluster to be shut down so as not to produce polysaccharide Paenan II is selected from the group consisting of a genetic modification that suppresses the function of glycosyltransferase PepT, pepU and/or PepV, and/or undecyipentenyl-glucose-phosphate transferase PepQ, and/or GDP-L-fucose synthase; and/or wherein the genetic modification that causes at least one enzyme function of the polysaccharide biosynthetic cluster to be turned off so as to not produce polysaccharide PAENAN III is selected from the group consisting of genetic modifications that suppress the function of glycosyltransferases PepI, pepJ, pepK and/or PepL.
6. The method of any one of claims 1 to 5, wherein the genetic modification that causes at least one enzyme function of the polysaccharide biosynthetic cluster to be turned off so as to produce neither polysaccharide Paenan II nor polysaccharide PAENAN III is selected from the group consisting of genetic modifications that suppress: the functions of glycosyltransferases PepI, pepT, pepU and PepV, or the functions of glycosyltransferases PepK, pepT, pepU and PepV, or the functions of glycosyltransferases PepL, pepT, pepU and PepV, or the functions of glycosyltransferases PepT and PepL.
7. The method of any one of claims 1 to 5, wherein the genetic modification that causes at least one enzyme function of the polysaccharide biosynthetic cluster to be turned off so as to produce neither polysaccharide Paenan I nor polysaccharide PAENAN III is selected from the group consisting of genetic modifications that suppress: the functions of glycosyltransferases PepF and PepJ, or glycosyltransferases PepD and PepJ.
8. The method of any one of claims 1 to 5, wherein the genetic modification that causes at least one enzyme function of the polysaccharide biosynthetic cluster to be turned off so as to produce neither polysaccharide Paenan I nor polysaccharide Paenan II is selected from the group consisting of genetic modifications that suppress: undecanopentenyl-glucose-phosphotransferase PepQ and glycosyltransferase PepF functions.
9. A composition comprising polysaccharide Paenan I, but neither polysaccharide Paenan II nor polysaccharide PAENAN III; or alternatively
A composition comprising polysaccharide Paenan II, but neither polysaccharide Paenan I nor polysaccharide PAENAN III; or alternatively
A composition comprising polysaccharide PAENAN III, but neither polysaccharide Paenan I nor polysaccharide Paenan II; or alternatively
A composition comprising polysaccharide Paenan I and polysaccharide Paenan II, but not comprising polysaccharide PAENAN III; or alternatively
A composition comprising polysaccharide Paenan II and polysaccharide PAENAN III, but not comprising polysaccharide Paenan I.
10. Is a Paenibacillus polymyxa producing organism in which at least one enzyme function of a polysaccharide biosynthetic cluster is turned off by genetic modification, thereby failing to produce polysaccharide Paenan II and polysaccharide PAENAN III; or alternatively
Is a Paenibacillus polymyxa producing organism in which at least one enzyme function of a polysaccharide biosynthetic cluster is turned off by genetic modification, thereby failing to produce polysaccharide Paenan I and polysaccharide PAENAN III; or alternatively
Is a Paenibacillus polymyxa producing organism in which at least one enzyme function of a polysaccharide biosynthetic cluster is turned off by genetic modification, thereby failing to produce polysaccharide Paenan I and polysaccharide Paenan II; or alternatively
Is a Paenibacillus polymyxa producing organism in which at least one enzyme function of a polysaccharide biosynthetic cluster is turned off by genetic modification, thereby failing to produce polysaccharide PAENAN III; or alternatively
Is a Paenibacillus polymyxa producing organism in which at least one enzyme function of a polysaccharide biosynthetic cluster is turned off by genetic modification, thereby failing to produce polysaccharide Paenan I.
11. Use of a polysaccharide comprising Paenan I, paenan II or PAENAN III or a mixture of any combination of Paenan I, paenan II and PAENAN III as a rheology, binder, stabilizer, emulsifier or flocculant, preferably in the food, pharmaceutical and/or cosmetic field.
12. Use according to claim 11 as an additive to food products, optionally selected from baked goods, soups, sauces, mayonnaise, ketchup, jams, citrus sauces, jellies, cans (fruits and vegetables), salad dressings, ice cream, mixed milk drinks, puddings, salted vegetables, canned meats and fish, or as an additive in cosmetic products, optionally selected from body washes, cosmetic creams and emulsions, shampoos, skin creams, toothpastes, moisturizing creams, or as an additive in pharmaceutical or medical products, optionally selected from wound dressings, granules for controlled release of active ingredient, eye drops, tablet coatings, capsules for nutritional supplements, or in tissue engineering to support surface adhesion after surgery, in water flooding for petroleum extraction, in bioremediation and waste water purification, for heavy metal binding, as a cement additive to keep granules suspended during curing, or as a coating additive.
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| PCT/EP2022/072461 WO2023036546A1 (en) | 2021-09-10 | 2022-08-10 | Polysaccharide or polysaccharide mixture produced by paenibacillus polymyxa |
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